Vitamin D derivative feed compositions and methods of use

ABSTRACT

Methods and compositions for enhancement of phytate phosphorus utilization and treatment and prevention of tibial dyschondroplasia in animals, particularly poultry, by administering to animals a feed composition containing a hydroxylated vitamin D 3  derivative. The vitamin D 3  derivative is preferably administered to animals in feed containing reduced levels of calcium and phosphorus for enhancement of phytate phosphorus utilization. The vitamin D 3  derivative is administered in combination with active phytase for further enhancement of phytate phosphorus utilization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.07/630,748 to Hardy Malcolm Edwards, Jr., for Treatment of TibialDyschondroplasia, filed Dec. 20, 1990, issued as U.S. Pat. No. 5,154,925Oct. 13, 1992, which is a continuation of U.S. patent application Ser.No. 07/311,540, filed Feb. 16, 1989, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the field of biochemistry, and particularlyrelates to animal feed compositions.

PHYTATE PHOSPHORUS UTILIZATION

Animals, including humans, require phosphorus in their diets for propergrowth and health. Farm animals are normally fed a grain-based animalfeed. Most of these grain-based feeds contain from 50-80% of their totalphosphorus as phytate phosphorus. Phytate phosphorus in plants occurs asthe mixed calcium-magnesium-potassium salt of the organic compound,phytic acid.

Many animals are unable to utilize most of the phytate phosphorus theyreceive in their feed. For example, studies by Edwards and Veltmann, J.Nutr. 113:1268-1575 (1983) and Ballam et al., Poultry Sci. 63:333-338(1984) with young broiler chickens fed corn-soybean diets indicatephytate phosphorus utilization of from only 10 to 53%. Feed consumed bythese animals must be supplemented with inorganic phosphorus, such as inthe form of dicalcium phosphate or defluorinated phosphate. The cost ofphosphorus supplementation is high. In addition, the unused phytatephosphorus is excreted, creating phosphorus soil contamination andcostly ecological problems.

The mechanisms involved in phytate phosphorus utilization by animals areunknown. Utilization of phytate phosphorus by chickens has been reviewedby several scientists including T. S. Nelson, Poultry Sci. 46:862-871(1967). Phytate phosphorus utilization in broiler chickens has beenshown by Edwards et al., Poultry Sci. 67:1436-1446 (1988) to beinfluenced by age. Other scientists, such as Lowe and Steenbock, BiochemJ. 30:1991-1995 (1936), Common, Agric. Sci. 30:113-131 (1940), Edwardsand Veltmann, J. Nutr. 113:1268-1575 (1983), Ballam et al., Poultry Sci.63:333-338 (1984) and Sooncharernying and Edwards, Poultry Sci.69(Suppl. 1):129 (1990), have shown that phytate phosphorus utilizationmay be influenced by calcium, phosphorus, and aluminum levels in thediet.

A method that would increase phytate phosphorus utilization by animals,especially farm animals such as chickens, would reduce the costs ofraising such animals because phosphate supplementation would beunnecessary. In addition, the soaring costs associated with thedecontamination of soil containing elevated phosphates would be greatlyreduced or even eliminated.

TIBIAL DYSCHONDROPLASIA

Tibial dyschondroplasia is a skeletal abnormality which occurs inrapidly growing animals such as broiler chickens or turkeys. The causeof tibial dyschondroplasia is unknown. Tibial dyschondroplasia isdistinguished from rickets, a vitamin D deficiency disease characterizedby overproduction and deficient calcification of osteoid tissue, in thata high incidence of tibial dyschondroplasia is found in animals thatreceive a sufficient dietary supply of vitamin D and are adequatelyexposed to sunlight.

Tibial dyschondroplasia is characterized by an unmineralized,unvascularized mass of cartilage located in the proximal ends of thetibiotarsus and the tarsusmetatarsus. The cartilage extends from thegrowth plate into the metaphysis. In fowl, tibial dyschondroplasiausually appears between three and eight weeks of age. In some chickensand turkeys, the prehypertrophic cartilage persists into adulthood butis restricted to the posterior medial portion of the proximaltibiotarsal bone so that the birds remain clinically normal. Anincidence of 10 to 30% of birds with subclinical dyschondroplasia iscommon in many flocks. In the more severe cases of tibialdyschondroplasia, the abnormal tissue occupies the whole metaphysis ofthe proximal tibiotarsal bone and also develops in the proximaltarsometatarsal bone.

Birds with these more severe lesions may be lame, with bowing of theaffected bones. These chickens are unable to walk normally and oftencollapse, causing injury and decreasing growth rate. The disease alsoincreases the death rate of animals during the growth period. Further,many of the birds suffering from tibial dyschondroplasia develop breastblisters and leg deformities that result in hemorrhages.

Tibial dyschondroplasia increases the percentage of carcasses that mustbe downgraded or condemned during processing, resulting in decreasedprofits for the processor. The deformed legs of birds with tibialdyschondroplasia often interfere with the shackling of the fowl duringprocessing and can actually cause mechanical problems in operating theprocessing line where the slaughtered fowl are conveyed on machineswhich handle the birds by their legs. Fowl with tibial dyschondroplasiahave insufficient leg strength to be carried in this manner.

A number of studies have been conducted to determine both the cause ofdyschondroplasia and a method for treatment or prevention. Leach andNesheim, "Further Studies on Tibial Dyschondroplasia CartilageAbnormality in Young Chicks", J. Nutr. 102:1673 (1972), indicated thatthe cartilage abnormality is a result of an inherited physiologicaldefect, the expression of which is under dietary control. They were notable to determine the nutritional factors responsible for expression.However, they found that manipulations of the mineral mixture thatresulted in changes in acid/base or cation/anion balance in the chickaltered the incidence of abnormality. In particular, high chloride levelin the diet increased the incidence of the abnormality.

Mongin and Sauveur, in "Interrelationship Between Mineral Nutrition,Acid-Based Balance, Growth and Cartilage Abnormalities," Growth andPoultry Meat Production, Borman, K. N. and Wilson, B. J., Eds., pp.235-247, British Poultry Science Ltd., Edinburgh, Scotland (1977),hypothesized that the metabolic acidosis in chickens fed high dietarychloride levels caused tibial dyschondroplasia because of impaired bonemineralization resulting from alteration of vitamin D metabolism.

In most animals, including humans, vitamin D₃ (cholecalciferol) ismetabolized first by hydroxylation at the C₂₅ position to25-hydroxycalciferol by one or more enzymes present in the liver, andthen hydroxylation of the 25-hydroxycalciferol at the C₁ position tocalcitriol (1,25-dihydroxycholecalciferol) by one or more enzymespresent in the kidneys. A flow chart showing the chemical structuresinvolved in vitamin D derivation and metabolism is shown in FIG. 1.

Chickens made acidotic by administration of ammonium chloride showreduced conversion of 25(OH)D₃ to 1,25(OH)₂ D₃, although the productionof 24,25-dihydroxycholecalciferol (24,25(OH)₂ D₃) from 25(OH)D₃ is notconsistently affected by acidosis as also reported by Sauveur and Monginin "Influence of Dietary Level of Chloride, Sodium and Potassium onChick Cartilage Abnormalities," Proceedings of XV World PoultryCongress, pp. 180-181 (1977).

However, supplementation of chickens with 20 ng/day of either1,25-dihydroxycholecalciferol (1,25(OH)₂ D₃) or24,25-dihydroxycholecalciferol (24,25(OH)₂ D₃) has been demonstrated tohave no effect on the incidence of tibial dyschondroplasia, as describedby Edwards in "Studies on the Etiology of Tibial Dyschondroplasia inChickens", J. Nutr., 114:1001 (1984).

Calcium and phosphorus levels in the diet have been found to be majornutritional factors influencing the expression of tibialdyschondroplasia. High calcium in the feed retards development of thelesion, whereas high phosphorus levels appear to accentuate thedevelopment of the lesions, as reported by Edwards and Veltmann, "TheRole of Calcium and Phosphorus in the Etiology of TibialDyschondroplasia in Young Chicks," J. Nutr., 113:1568 (1983).

Increases in the magnesium content of the chick diet decrease theincidence of tibial dyschondroplasia; however, the effect of magnesiumis not as strong as that of calcium, as demonstrated by Edwards,"Studies on the Etiology of Tibial Dyschondroplasia in Chickens", J.Nutr., 114:1001 (1984).

Given the large economic loss to meat producers caused by animalsafflicted with tibial dyschondroplasia as well as the discomfort of theafflicted animal and the resulting unsanitary conditions caused by thediseased dysfunctional animal, it would be of great benefit to find aneffective method and compositions to reduce the incidence of thisdisease.

U.S. Pat. No. 5,154,925, of which this is a continuation-in-part,discloses a method for treating tibial dyschondroplasia in fowl andother animals by administering to the animals a vitamin D₃ derivativeincluding 1,25-dihydroxycholecalciferol;1,25-dihydroxy-26,27-hexadeuterocholecalciferol;1-hydroxycholecalciferol; 1,24,25-trihydroxycholecalciferol;1,25-dihydroxy-24-fluorocholecalciferol; 25-hydroxycholecalciferol, orcombinations thereof. The derivative is administered or fed to animalsin a pharmaceutical carrier including animal feed in a concentrationbetween 0.10 and 20 micrograms of a vitamin D₃ derivative per kilogramof body weight per day. U.S. Pat. No. 5,154,925 has claims directed toadministration of the vitamin D₃ derivatives1,25-dihydroxy-26,27-hexadeuterocholecalciferol;1,24,25-trihydroxycholecalciferol; and1,25-dihydroxy-24-fluorocholecalciferol to fowl and other animals in apharmaceutically acceptable carrier including animal feed.

It is therefore an object of the present invention to provide additionalmethods and compositions for the treatment and prevention of tibialdyschondroplasia in animals.

It is a further object of the present invention to provide methods andcompositions for enhancing phytate phosphorus utilization in animals.

It is a further object of the present invention to provide a method andcompositions for enhancing phytate phosphorus utilization whilepreventing or treating tibial dyschondroplasia.

It is further object of the present invention to provide methods andcompositions for decreasing phosphorus contamination of soil by animals.

It is a further object of the present invention to provide economicalanimal feed compositions containing reduced levels of supplementalcalcium and phosphorus.

SUMMARY OF THE INVENTION

The methods described herein include feeding animals a feed compositioncontaining an effective amount of a hydroxylated metabolite, orderivative, of vitamin D₃, including 1,25-dihydroxycholecalciferol;1,25-dihydroxy-26,27-hexadeuterocholecalciferol;1-hydroxycholecalciferol; 1,24,25-trihydroxycholecalciferol;1,25-dihydroxy-24-fluorocholecalciferol; 25-hydroxycholecalciferol, orcombinations thereof, in animal feed. Feed compositions containing anamount of vitamin D₃ derivative effective for enhancing phytatephosphorus utilization and treatment or prevention of tibialdyschondroplasia contain between 1 and 10 micrograms derivative perkilogram feed.

It has been discovered that feeding a feed composition containing one ormore of the above-defined 1-hydroxy vitamin D₃ derivatives increasescalcium utilization, so less dietary calcium is needed. A reduction indietary calcium enhances phytate phosphorus utilization so that lesssupplemental phosphorus is required in the diet. Concurrentadministration of phytase further enhances phytate phosphorusutilization.

For prevention of tibial dyschondroplasia, a feed composition containingbetween 1 and 10 micrograms of one or more of the above-definedhydroxylated vitamin D₃ derivatives per kilogram of feed is fed on thefirst day of life and is continued for at least three weeks. Treatmentof tibial dyschondroplasia already established in poultry is achieved byfeeding the feed composition as soon as the disease is discovered andcontinuing treatment until the animal is cured.

BRIEF DESCRIPTION OF THE FIGURERS

FIG. 1 is a drawing of the chemical structures of the synthesis ofvitamin D₃ from 7-dehydrocholesterol to previtamin D₃ to vitamin D₃ andthe metabolism of vitamin D₃ to 1,25-dihydroxycholecalciferol.

FIG. 2 is a drawing of the chemical structures of vitamin D₃(cholecalciferol) (1), 1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5),1,25-dihydroxy-24-fluorocholecalciferol (6), and25-hydroxycholecalciferol (7) as used in the methods and compositionsdescribed herein.

FIG. 3 is a drawing of the chemical structure of24,25-dihydroxycholecalciferol (8).

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for enhanced phytate phosphorus utilization andthe treatment and prevention of tibial dyschondroplasia in animals areprovided herein. Enhancement of phytate phosphorus utilization resultsin more economical food production by reducing the need for dietarycalcium and phosphorus supplementation and reducing the amount ofenvironmentally-hazardous phosphate excreted by the animal. Treatment orprevention of tibial dyschondroplasia improves the health of the animalsand the sanitary conditions of the animal facility, and allows moreanimals to be successfully processed before and after slaughter.

ENHANCEMENT OF PHYTATE PHOSPHORUS UTILIZATION

A method for enhancement of phytate phosphorus utilization is providedthat includes feeding to animals an animal feed composition containingan effective amount of a 1-hydroxy vitamin D₃ derivative including1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5),1,25-dihydroxy-24-fluorocholecalciferol (6), and combinations thereof.

Because the composition increases the utilization of calcium from thediet, the diet, such as an animal feed diet, contains lower levels ofcalcium without risk of adverse consequences such as bone deformity orbreakage. Preferably, the total concentration of calcium in the diet isbetween 0.5 and 1.3% by weight. Most preferably, the calciumconcentration is less than 1%.

The reduced calcium levels cause enhancement of phytate phosphorusutilization allowing a reduction in the amount of supplementalphosphorus supplied in the diet and a reduction in the amount ofphosphorus excreted by the animal. Preferably, the total concentrationof phosphorus in the diet is between 0.4 and 0.9% by weight.

The composition can additionally include phytase to enhance phytatephosphorus utilization to an even greater extent. Phytase is an enzyme,normally isolated in large quantities from E. coli or Asperigillusficuum, that converts phytate phosphorus to inorganic phosphate ions.Preferably, active phytase is added to the composition by spraying feedwith an effective amount of a solution of active phytase after the feedhas been formed into pellets to avoid inactivation of the enzyme byexposure to elevated temperatures during the pelleting process. Mostpreferably, the feed contains between 30 and 600 units of active phytaseper kilogram feed.

PREVENTION OR TREATMENT OF TIBIAL DYSCHONDROPLASIA

A method for the treatment or prevention of tibial dyschondroplasia isprovided that includes feeding animals an animal feed compositioncontaining an effective amount of a 1-hydroxy vitamin D₃ derivativeincluding 1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5),1,25-dihydroxy-24-fluorocholecalciferol (6), 25-hydroxycholecalciferol(7), and combinations thereof.

PREPARATION OF VITAMIN D₃ DERIVATIVES

The vitamin D₃ derivatives described herein can be prepared by thefollowing procedures, the teachings of which are incorporated byreference herein.

1,25-Dihydroxycholecalciferol can be prepared as described inBiochemistry 10(14), 2799 (1971), and U.S. Pat. Nos. 4,310,467 and3,697,559.

1,25-Dihydroxy-26,27-hexadeuterocholecalciferol can be prepared asdescribed for the synthesis of 1,25-dihydroxycholecalciferol in Tet.Let. 40, 4147 (1972), with the substitution of a trideuteromethylGrignard reagent in place of the methyl Grignard reagent used to add thecarbons at the 26 and 27 positions.

1-Hydroxycholecalciferol can be prepared by the methods described in J.Am. Chem. Soc. 95(8), 2748 (1973) and U.S. Pat. No. 3,741,996.

1,24,25-Trihydroxycholecalciferol can be prepared by the methoddescribed in U.S. Pat. No. 3,847,955.

1,25-Dihydroxy-24-fluorocholecalciferol can be prepared by the proceduredescribed in J. Org. Chem., 53(5), 1040 (1988).

25-Hydroxycholecalciferol can be prepared as described in U.S. Pat. Nos.4,310,467 and 3,565,924.

ANIMALS TO WHICH THE COMPOSITION IS ADMINISTERED

The methods and compositions described herein are particularly usefulfor enhancing phytate phosphorus utilization and reducing the incidenceof tibial dyschondroplasia in chickens, especially broiler chickens andegg-laying chickens.

The methods and compositions are equally effective in enhancing phytatephosphorus utilization in other types of monogastric animals, includingbut not limited to, humans, swine, and other fowl including turkeys,pheasants, and ducks. The animals to which the animal feed containingthe vitamin D₃ derivative is fed are generally not vitamin D-deficient,receiving an adequate supply of vitamin D in the diet and exposure toadequate amounts of sunlight.

Feeding the above-described feed compositions according to the methodsdescribed herein are also effective in preventing or treating tibialdyschondroplasia by reducing or reversing the development of abnormalcartilage at the proximal end of the tibia in a variety of animalsincluding, but not limited to, swine, dogs, rabbits, cattle, fish andother fowl including turkeys, pheasants and ducks. For example, GreatDanes have a particular problem with tibial dyschondroplasia due totheir rapid growth.

EFFECTIVE DOSAGE RANGE AND METHOD OF ADMINISTRATION

An effective dosage of one or more of the vitamin D₃ derivativesdescribed herein for either enhancement of phytate phosphorusutilization or prevention or treatment of tibial dyschondroplasia isbetween 0.1 and 20 micrograms per kilogram of body weight per day.However, it is inconvenient to weigh the animal and adjust andadminister the dosage each day. It is more convenient and economical toprepare an animal feed or water composition containing a fixed amount ofvitamin D₃ derivative. As the animal becomes larger, it will consumegreater quantities of feed or water containing the vitamin D₃derivative, thereby self-regulating administration of an appropriatedosage of the derivative.

For the foregoing reasons, the vitamin D₃ derivative is preferably fedto animals in an animal feed composition containing between 1 and 10micrograms derivative per kilogram feed. A more preferred animal feedcomposition contains between 3 and 6 micrograms derivative per kilogramfeed. Most preferably, the animal feed composition is fed in feedcontaining approximately 5 micrograms vitamin D₃ derivative per kilogramfeed. Dosages above 10 micrograms derivative per kilogram feed tend tocause a decreased animal growth rate.

It will be understood by those skilled in the art that the vitamin D₃derivative could also be administered as a water composition wherein theconcentration of derivative in the water is approximately one-half theconcentration of derivative described above for use in feed becauseanimals normally consume two volumes of water for every one volume ofdry food. Therefore, the concentration of vitamin D₃ derivative in wateris preferably between 0.5 and 5 micrograms per kilogram water.

Enhancement of both phytate phosphorus utilization and prevention oftibial dyschondroplasia can be achieved by feeding one or more of thevitamin D₃ derivatives in accordance with the methods described hereinin animal feed from birth throughout the life of the animal. Forexample, in poultry, treatment is preferably begun at one day of age,and continued for at least three to four weeks to prevent the onset oftibial dyschondroplasia. Feeding the vitamin D₃ derivatives describedherein to animals older than one day of age will continue to provide thedesired therapeutic effect, but will be slightly less effective atpreventing the onset of the tibial dyschondroplasia.

The compositions can also be fed in a feed composition as a treatmentfor tibial dyschondroplasia already established in the animal. The samedosages are used as for prevention of the disease. The compositionsshould be fed to the animal as soon as the disease is discovered, andcontinued until the animal is cured. However, feeding the feedcompositions at this time will not reverse the abnormal shape of bonescaused by the disease.

The effectiveness of feeding the composition on enhancement of phytatephosphorus utilization in poultry and other animals can be determined bymeasuring the phosphorus content of the feed, the phosphorus content offeces collected from the animals, and calculating the amount ofphosphorus retained as described in the examples below.

The effectiveness of feeding the compositions on prevention or treatmentof tibial dyschondroplasia in fowl can be determined by making alongitudinal cut across the tibia and inspecting it for incidence andseverity of the disease, as described below in the examples.

The effectiveness of feeding the compositions containing the vitamin D₃derivatives on prevention or treatment of dyschondroplasia in otheranimals can be determined by internal inspection of the bone forabnormal cartilage development or external inspection of bone forirregularities, bowed shape, or weakness.

POULTRY FEED COMPOSITION FOR PHYTATE PHOSPHORUS ENHANCEMENT

A preferred poultry feed composition for enhancement of phytatephosphorus utilization in poultry contains an effective amount of a1-hydroxylated vitamin D₃ derivative such as1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5),1,25-dihydroxy-24-fluorocholecalciferol (6), or a combination thereof,in combination with sources of energy such as corn, fat and soybeanmeal; sources of protein such as corn and soybean meal; mineral sourcessuch as iodized sodium chloride; amino acids such as D,L-methionine; avitamin mixture; a trace mineral mixture; selenium concentrate (0.02%sodium selenite); and a calcium source such as limestone.

The vitamin mixture in the poultry or fowl feed composition provides inmilligrams/kilogram of diet (except as noted) vitamin A (asall-trans-retinyl acetate), 5,500 IU; vitamin D₃ (cholecalciferol), 1100ICU or 27.5 micrograms; vitamin E (all-rac-alpha-tocopheryl acetate), 11IU; riboflavin, 4.4; calcium pantothenate, 12; nicotinic acid, 44;choline chloride, 220; vitamin B₁₂, 9 micrograms; vitamin B₆, 3.0;menadione (as menadione sodium bisulfite), 1.1; thiamin (as thiaminmononitrate) 2.2; folic acid, 3; biotin, 0.3; and ethoxyquin, 125.

The trace mineral mixture provides in milligrams per kilogram of dietMnO₂, 222; ZnO, 150, FeSO₄ -7H₂ O, 200; FeCO₃, 83; C_(u) SO₄, 29; andCa(IO₃)₂, 15.

The vitamin D₃ derivative is preferably combined with the feed by firstdissolving it in an alcohol, adding the solution to feed, and mixing thefeed to evenly distribute the dissolved derivative.

A phytase solution is preferably added to the feed by first diluting thephytase with a powder, such as powdered soybean meal flour and thenmixing the diluted phytase with the feed composition containing thevitamin D₃ derivative. Alternatively, the phytase solution can besprayed onto the feed and allowed to dry. Preferably, the feed containsbetween 30 and 600 units of active phytase per kilogram feed. Activephytase is available commercially from the Sigma Chemical Company, St.Louis, Mo.

The ingredients of a supplemented chicken feed to which the vitamin D₃derivatives described herein may be added in the above-described dosagesfor enhanced phytate utilization in chickens are described in Table 1.The amounts indicated are given in percent by weight. It will beunderstood by those skilled in the art that the active vitamin D₃derivatives described herein can also be fed in combination with othercommercially formulated or similar feeds for chickens and other animals.

                  TABLE 1                                                         ______________________________________                                        Chicken Feed Composition for Phytate                                          Phosphorus Enhancement                                                        Ingredients              Amounts                                              ______________________________________                                        Ground yellow corn       57.63                                                Soybean meal (dehulled)  35.00                                                Poultry fat (stabilized) 5.00                                                 Iodized sodium chloride  0.45                                                 D,L-Methionine (98%)     0.20                                                 Vitamin premix (as described above)                                                                    0.25                                                 Trace mineral premix (as described above)                                                              0.10                                                 Se concentrate (0.02% from sodium selenite)                                                            0.05                                                 Limestone                1.32                                                 ______________________________________                                    

The vitamin D₃ derivatives described herein are efficacious for theenhancement of phytate phosphorus utilization when added to theabove-described feed which has a calcium concentration of 0.5%. Broilerchickens are typically fed a diet which has a range of calcium of 0.7 to1.4% by weight, while egg laying hens are fed a diet with a highercalcium level. This reduced level of calcium is achieved because theanimal feed contains no dicalcium phosphate. Any calcium present in thefeed is due to the presence of limestone.

A reduced level of phosphorus of 0.3% is also achieved by feeding theabove-described animal feed lacking dicalcium phosphate. The phosphatepresent is due to phytate phosphorus alone.

POULTRY FEED COMPOSITION FOR TIBIAL DYSCHONDROPLASIA

A preferred poultry feed composition for treatment or prevention oftibial dyschondroplasia in poultry contains an effective amount of ahydroxylated vitamin D₃ derivative such as 1,25-dihydroxycholecalciferol(2), 1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5),1,25-dihydroxy-24-fluorocholecalciferol (6), 25-hydroxycalciferol (7),or a combination thereof, in combination with sources of energy such ascorn, fat and soybean meal; sources of protein such as corn and soybeanmeal; mineral sources such as iodized sodium chloride; amino acids suchas D,L-methionine; a vitamin mixture; a trace mineral mixture; seleniumconcentrate (0.02% sodium selenite); a phosphorus source such asdicalcium phosphate; and calcium sources such as dicalcium phosphate andlimestone.

The vitamin and trace mineral mixtures for the chicken feed compositionfor the treatment or prevention of tibial dyschondroplasia are the sameas those set forth above for the chicken feed composition forenhancement of phytate phosphorus utilization.

The ingredients of a supplemented chicken feed to which the vitamin D₃derivatives described herein may be added in the above-described dosagesfor treatment or prevention of tibial dyschondroplasia in chickens aredescribed in Table 2. The amounts indicated are given in percent byweight. It will be understood by those skilled in the art that theactive vitamin D₃ derivatives described herein can also be fed incombination with other commercially formulated or similar feeds forchickens and other animals.

                  TABLE 2                                                         ______________________________________                                        Chicken Feed Composition for Tibial Dyschondroplasia                          Ingredients              Amounts                                              ______________________________________                                        Ground yellow corn       56.81                                                Soybean meal (dehulled)  35.00                                                Poultry fat (stabilized) 5.00                                                 Iodized sodium chloride  0.45                                                 D,L-Methionine (98%)     0.20                                                 Vitamin premix (as described above)                                                                    0.25                                                 Trace mineral premix (as described above)                                                              0.10                                                 Se concentrate (0.02% from sodium selenite)                                                            0.05                                                 Dicalcium phosphate (feed grade)                                                                       1.86                                                 Limestone                0.28                                                 ______________________________________                                    

Chickens fed the diet described in Table 2 without the vitamin D₃derivatives described herein will have a higher than normal incidence oftibial dyschondroplasia even though the diet contains a high level ofvitamin D₃ because the diet has a high level of chloride and phosphorusand a low level of calcium. The average analyzed values for the chickenfeed described in Table 2 are 0.32% chloride, 0.76% phosphorus, and0.75% calcium.

The vitamin D₃ derivatives described herein are efficacious for thetreatment or prevention of tibial dyschondroplasia in a feed having anycalcium concentration, particularly those with a calcium composition ofless than 2.5% by weight. However, a feed composition useful for bothenhancement of phytate phosphorus utilization and prevention ortreatment of tibial dyschondroplasia preferably has a calciumconcentration of between 0.5 and 1.3% as described above.

A control system to test the effectiveness of the vitamin D₃ derivativeson reducing the incidence of tibial dyschondroplasia in fowl wasdeveloped using disulfiram [bis(diethylthiocarbamyl)disulfide], analcohol deterrent, in a dietary dosage of approximately 30 mg/kg feedper day. Disulfiram supplementation lowers the absorption of calciumwhen given as an oral dose compared to controls. It is known thatthiruram [bis(dimethylthiocarbamyl)disulfide], a compound used as afungicide and bactericide which is structurally close to disulfiram andwhich also causes tibial dyschondroplasia, creates a rapid loss ofintramuscularly-injected calcium immediately after dosing. This suggeststhat calcium in the blood and soft tissues (gastro-intestinal tract) canbe lost more easily from the birds receiving thiruram or disulfiram butthat once the calcium goes into the bone both thiruram and disulfiramhave little effect on turnover.

The present methods and compositions will be further understood withreference to the following non-limiting examples.

EXAMPLE 1: ENHANCEMENT OF PHYTASE UTILIZATION WITH VITAMIN D₃DERIVATIVES

The effectiveness of 1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5), and1,25-dihydroxy-24-fluorocholecalciferol (6) in enhancing phytatephosphorus utilization in chickens has been demonstrated using thefollowing experimental procedures.

Newly hatched Petersen X Arbor Acre cockerels obtained from a commercialhatchery were used. Animals were housed in electrically heated Petersimebattery brooders with wire-mesh floors located in a room in which thetemperature was maintained at 22° C., fluorescent lights were on 24hours each day in the room and the cages, and sunlight was presentduring the day from large windows. No attempt was made to limit thenatural production of vitamin D₃. Feed and water were available to thechickens at all times.

Ten newly hatched birds were randomly placed in each section of thePetersime batteries until the birds were nine days old. The measurementsdescribed below were calculated on a pen basis; all statistical analyseswere conducted on pen averages. Blood samples were taken by cardiacpuncture with heparinized syringes from one randomly chosen bird fromeach pen on the ninth day before weighing the birds. The blood sampleswere analyzed for plasma total calcium using Technicon Auto AnalyzerMethodology set forth in a 1969 publication by the Technicon InstrumentCo. Ltd. (Basingstoke, Hants, U.K.) and dialyzable phosphorus usingTechnicon Auto Analyzer Methodology set forth in a 1970 publication bythe Technicon Instrument Co. Ltd.

The basal diet used in these studies is shown in Table 1 above. Thebasal diet contained 1100 ICU/kg (27.5 microgram/kg) of added D₃ and islow in calcium and phosphorus, having average analyzed values of 0.5%calcium and 0.3% phosphorus.

At nine days of age, pen body weights were obtained for all birds andtheir feed consumption was recorded. All birds were then killed byasphyxiation and examined at random for tibial dyschondroplasia andrickets by making a longitudinal cut across the tibia and scoring forincidence and severity of tibial dyschondroplasia in accordance with themethod described in H. Edwards and J. Veltmann, "The Role of Calcium andPhosphorus in the Etiology of Tibial Dyschondroplasia in Young Chicks",J. Nutr., 113, 1568 (1983), which is incorporated herein by reference.Briefly, using a three week old chicken as the reference animal, a scoreof zero indicates normal cartilage which is narrow with littleirregularities. A score of one indicates cartilage which is thickened orshows considerable irregularities. A score of two indicates that thecartilage is thickened and there is evidence of persistingprehypertrophic cartilage that is not calcified and which has not beeninvaded by vessels from the metaphysis. Deep irregularities areapparent. A score of three indicates a large mass of cartilage in theproximal end of the tibia. The birds were also scored for phosphorusdeficient rickets at the same time they were scored for tibialdyschondroplasia. Chickens which had normal proliferatingprehypertrophied zone and a lengthened metaphyseal primary spongiosawere classified as phosphorus deficient rickets. The left tibia wasremoved for bone ash determination on the fat-free bone.

In each experiment, the droppings from all of the chickens for theentire nine day experimental period were collected at the end of theexperiment. They were dried in a force draft oven at 70° C., weighed,and ground in a Wiley Mill. Samples were then taken and, with a feedsample, analyzed for calcium by the fluorometric method of Hill, Clin.Chem. 2:122-130 (1955), phosphorus by the method of O'Neill and Webb, J.Sci Food Agric. 21:217-219 (1970), and phytate, or phytin, phosphorus bythe method of Common, J. Agric. Sci. 30:113-131 (1940), the methods ofwhich are incorporated by reference herein.

Phytate phosphorus in feed and feces samples was also determined by amodification of the method reported by Sandberg and Ahderinne, J. FoodSci. 51:547-550 (1986), the methods of which are incorporated byreference herein, for determination of phytate in feed and feedingredients. The method has been modified and tested to allow highpressure liquid chromatography determination of phytate in poultry fecesas follows. Pulverized dried excreta of 0.2 g (0.5 g for feed sample)was extracted using 20 ml 0.5M HCl under vigorous mechanical agitationat room temperature for two hours. The extract was filtered throughglass microfiber filter paper #934-AH (Whatman International, Maidstone,England) under vacuum. Four milliliters of the filtrate was then dilutedwith 16 ml of deionized water. The inositol phosphates were separatedfrom the diluted filtrate solution and were concentrated usinganion-exchange column chromatography. A glass column of 0.7×15 cm with aglass filter at the distal end was packed with 2 ml of anion-exchangeresin (AG 1-x8, 200-400 mesh) (Bio-Rad, Richmond, Calif.) developedovernight in 0.025M HCl.

The diluted filtrate solution of 20 ml was passed through theanion-exchange column at a flow rate of approximately 0.4 ml/min, thecolumn was then washed with 4 ml 0.025M HCl. Inositol phosphates wereremoved from the chromatographic column using 20 ml 2M HCl. The eluatewas collected in a 20 ml scintillation vial. An 8 ml fraction of eluentwas freeze-dried.

The dried sample was then reconstituted with 0.8 ml of a filtered mobilephase consisting of 0.05M formic acid:methanol (HPLC grade) 50:50, 1.5ml/100 ml of tetrabutylammonium hydroxide (TBA-OH, 40% in water) (Sigma,St. Louis, Mo.) and 5 mM of ethylenediaminetetraacetic acid (EDTA). ThepH of the mobile phase was adjusted to 4.3 by the addition of 6Msulfuric acid. The mobile phase was filtered through a nylon membranefilter (0.45 micron pore size) under vacuum.

The analysis was performed using an HPLC pump, Eldex Model AA-72-S(Eldex, San Carlos, Calif.) equipped with a reverse phase C-18 WatersRadialpak column (Waters, Milford, Mass.) having a 5 mm inside diameterand 5 micron particle size. The inositol phosphates were detected byrefractive index using a Waters Differential Refractometer, Model R401(Waters, Milford, Mass.). The optimal flow rate was 1.5 ml/min.Retention times and peak areas were measured by a Hewlett Packard Model3390A integrator (Hewlett Packard, Palo Alto, Calif.). Samples wereinjected into a 20 microliter Rheodyne Model 7125 loop (Rheodyne,Cotati, Calif.). Sodium phytate was used as both the internal andexternal standard. The linearity of phytate concentration versus peakarea was investigated by 20 microliter injections of solutionscontaining 5.6, 11.2, 16.8, and 22.4 micrograms of phytate.

In the first experiment, a sample containing a known amount of addedhexaphosphoinositol was run with each sample to determine recovery. Forthe 24 fecal samples in this experiment, the recovery averaged 101.5%.Recovery studies were not conducted in later experiments and recoverywas assumed to be 100%.

EXPERIMENT 1

Each of the twelve different dietary treatments described below wereprovided to two pens containing ten birds per pen.

1. One diet was a basal diet lacking supplemented inorganic phosphorus,phytase or the vitamin D₃ derivative 1,25-(OH)₂ D₃ (2).

2. Three diets contained graded levels of phosphorus (0.1, 0.2 and 0.3%)from reagent grade Na₂ HPO₄ H₂ O which is indicated by the symbol P inthe tables.

3. Four diets contained graded levels of phytase (Sigma Chemical Co.,St. Louis, Mo.), at 75, 150, 300 and 600 units/kg of feed. The phytasewas first mixed with powdered soybean meal flour, and the phytase andflour mixture was combined with the feed.

4. Four diets contained the four levels of phytase described above plus10 micrograms/kg of 1,25-(OH)₂ D₃. The derivative was first diluted withpropylene glycol to a concentration of 10 micrograms per milliliter, andthen the solution was mixed with feed in a Hobart mixer for 10 minutes.

All chickens were fed a corn-soybean diet containing only 0.67% calcium,0.34% phosphorus, and a vitamin mixture supplying 1100 ICU D₃ /kg ofdiet.

The results are shown in Tables 3A, 3B and 3C below.

ANALYSIS OF EXPERIMENT 1 RESULTS

The birds fed the basal phosphorus-deficient diet had very low nine dayweights and gain to feed ratios and developed very severe phosphorusdeficient rickets. The bone ash value of 22.9% ash indicates extremelypoor bone calcification. Fifty percent of the dietary phytate appearedin the feces as hexa, hepta and tetra phosphoinositol, (IP6, IP5 andIP4) indicating that only half of the phytate phosphorus in the basaldiet was available to the chickens.

The addition of phosphorus to the diet resulted in increased nine dayweights, gain:feed ratios, blood plasma phosphorus and bone ash. Theincidence of phosphorus-deficient rickets was decreased. The amount ofphosphorus in the feces was approximately the same.

The addition of phytase to the diet resulted in an increase in nine dayweight and a small decrease in the amount of hexaphosphoinositol in thefeces. There was no significant increase in bone ash. The calculatedphytate retention value was 60.7% for birds fed the highest level ofphytase.

The animals given feed containing 1,25-(OH)₂ D₃ (2) and phytase hadhigher nine day body weights than those fed the basal diet. The additionof 1,25-(OH)₂ D₃ and phytase to the diet resulted in increased bone ashvalues and a significant decrease in the levels of hexa and pentaphosphoinositol in the feces as compared to all other treatments. Thelevel of tetraphosphoinositol was so low in feces that it was notdetectable with the method used. The calculated retention data indicatethat these animals utilized from 76 to 88% of the phytate phosphorusconsumed as compared to 50% by the animals fed the basal diet and 52 to60% by the animals fed the basal diet plus phytase alone.

The addition of 1,25-(OH)₂ D₃ caused a large increase in the utilizationof phytate phosphorus as indicated from the bone ash values and amountsof phytate excreted in the feces.

                                      TABLE 3A                                    __________________________________________________________________________    Effect of dietary phosphorus levels and phytase and/or 1,25-(OH).sub.2        D.sub.3 supplementation on growth,                                            developement of tibial dyschondroplasia and Ca, P, and                        phytate phosphorus retention by broilers                                                               Tibial dyschondroplasia                                                                     Phosphorus                                                                          Retention by the broiler                     Wt.      Bone         #3   rickets          Phytate                           9-d Gain:                                                                              ash Incidence                                                                              Scores                                                                             incidence                                                                           Calcium                                                                            Phosphorus                                                                          phosphorus.sup.1      Treatments  g   feed %   %    Score                                                                             %    %     %    %     %                     __________________________________________________________________________    Basal 0% P  133 .672 22.9                                                                              12   2.00                                                                              7    41    49.53                                                                              46.47 50.50                 .1% P       168 .753 21.8                                                                              35   1.80                                                                              10   5     52.76                                                                              40.72 46.51                 .2% P       164 .701 36.5                                                                              15   .67 0    0     60.87                                                                              28.56 67.78                 .3% P       163 .701 38.5                                                                              5    .50 0    0     62.20                                                                              9.50  50.72                  75 units Phytase                                                                         144 .724 23.9                                                                              0    0   0    33    47.06                                                                              47.2  56.36                 150 units Phytase                                                                         148 .716 24.7                                                                              5    .50 0    28    44.80                                                                              53.55 59.06                 300 units Phytase                                                                         142 .707 24.4                                                                              0    0   0    40    42.62                                                                              49.04 52.67                 600 units Phytase                                                                         153 .733 26.5                                                                              25   1.63                                                                              10   30    43.54                                                                              55.39 64.85                  75 units P +                                                                             162 .727 31.7                                                                              0    0   0    5     51.14                                                                              61.73 80.60                 10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           150 units P +                                                                             152 .700 32.1                                                                              5    .50 0    0     51.15                                                                              67.24 87.41                 10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           300 units P +                                                                             153 .690 33.5                                                                              5    .50 0    5     56.80                                                                              65.50 83.36                 10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           600 units P +                                                                             160 .729 34.0                                                                              5    .50 0    0     53.50                                                                              67.31 82.23                 10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           Mean ± SEM                                                                             153 ±                                                                          .713 ±                                                                          30.0 ±                                                                          9 ±                                                                            .72 ±                                                                           2 ±                                                                             15 ±                                                                            51.33 ±                                                                         49.35                                                                               65.17 ±                         5  .016 .9  8    .51 3    8     3.29 1.60  3.99                  __________________________________________________________________________     .sup.1 Total feed and fecal phytate phosphorus determined by the ferric       chloride method.                                                         

                                      TABLE 3B                                    __________________________________________________________________________    Effect of dietary phosphorus levels and phytase and/or 1,25-(OH).sub.2        D.sub.3 supplementation on blood Ca and P,                                    phytin phosphorus excretion, and calculated retention of phytate              phosphorus                                                                                           Phytin phosphorus excretion expressed as                                                           Phytin Phosphorus                             Blood plasma                                                                             percent of total phytin phosphorus                                                                 retained                                           Dialyzable            Total                                                                              (100 - total                                  Calcium                                                                            phosphorus                                                                          IP4  IP5  IP6   excreted                                                                           excreted)                         Treatments  mg/kg                                                                              mg/kg %    %    %     %    %                                 __________________________________________________________________________    Basal 0% P  136  20.0  .25  5.20 44.83 50.4 49.8                              .1% P       142  20.5  .71  5.76 46.92 53.4 46.7                              .2% P       112  59.5  .81  2.70 37.06 40.6 59.5                              .3% P       105  75.0  1.46 6.87 46.00 54.4 45.9                               75 units Phytase                                                                         137  15.8  .28  5.05 43.12 48.4 51.6                              150 units Phytase                                                                         145  14.8  .00  4.23 37.46 41.7 58.5                              300 units Phytase                                                                         161  7.67  .39  5.14 40.66 46.2 53.8                              600 units Phytase                                                                         145  1.83  .43  4.48 34.46 39.4 60.7                               75 units P +                                                                             168  18.0  .00  1.28 22.43 23.8 76.3                              10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           150 units P +                                                                             178  14.0  .00  1.12 19.53 20.7 79.4                              10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           300 units P +                                                                             157  24.0  .000 .61  12.91 13.5 86.5                              10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           600 units P +                                                                             148  25.0  .000 .13  12.07 12.3 87.8                              10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           Mean ± SEM                                                                             144 ±                                                                           24.7 ±                                                                           .36 ±                                                                           3.55 ±                                                                          33.12 ±                                                                          37.0 63.0 ±                                      14  6.6   .05  .75  2.54  3.1  3.1                               __________________________________________________________________________

                                      TABLE 3C                                    __________________________________________________________________________    Phytate content of fecal samples, determined by the ferric chloride and       HPLC procedures                                                               and calculated retention of phytate phosphorus by each procedure                                     Phytate                                                                       Phosphorus                    Phytate                              Food.sup.1                                                                          Feces                                                                              in feces by                                                                         Phytate phosphorus in feces by HPLC                                           procedure               phosphorus                           consumed                                                                            excreted                                                                           FeCl.sup.3                                                                          IP4   IP5   IP6   Total retention                Treatments  g     g    mg/g  mg/g  mg/g  mg/g  mg/g  FeCl.sup.3                                                                         HPLC                __________________________________________________________________________    Basal 0% P  1345  402  3.53  0.25  .490  4.23  4.75  51   50                  .1% P       1660  451  3.50  .060  .490  3.99  4.54  47   47                  .2% P       1725  602  1.97  .065  .230  3.03  3.32  68   60                  .3% P       1715  606  2.98  .115  .550  3.68  4.35  51   46                   75 units Phytase                                                                         1395  460  2.87  .025  .435  3.73  4.19  56   52                  150 units Phytase                                                                         1305  434  2.63  .000  .360  3.18  3.54  59   59                  300 units Phytase                                                                         1385  483  2.93  .030  .410  3.23  3.68  53   54                  600 units Phytase                                                                         1500  491  2.30  .035  .385  2.96  3.38  65   61                   75 units P +                                                                              475  519  1.17  .000  .105  1.80  1.90  81   76                  10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           150 units P +                                                                             1565  564  0.76  .000  .090  1.53  1.62  87   79                  10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           300 units P +                                                                             1510  522  1.03  .000  .050  1.06  1.11  83   87                  10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           600 units P +                                                                             1610  561  1.09  .000  .010  0.98  0.99  82   88                  10 μg/kg 1,25-(OH).sub.2 D.sub.3                                           Mean ± SEM                                                                             1515 ±                                                                           508 ±                                                                           2.23 ±                                                                           .030 ±                                                                           .300 ±                                                                           2.78 ±                                                                           3.11 ±                                   96    50  .28   .015  .067  .27   .33                            __________________________________________________________________________

EXPERIMENT 2

Each of the eight different dietary treatments set forth below wereprovided to three pens containing ten birds per pen.

1. One diet was a basal diet lacking supplemental inorganic phosphorus,phytase or the vitamin D₃ derivative 1,25-(OH)₂ D₃ (2).

2. One diet was a basal diet contained 0.2% supplemental phosphorus (Na₂HPO₄ H₂ O) alone.

3. One diet contained 75 units of phytase alone.

4. One diet contained 5 micrograms/kg 1,25-(OH)₂ D₃ alone.

5. One diet contained both 0.2% supplemental phosphorus plus 75 units ofphytase.

6. One diet contained both 0.2% supplemental phosphorus plus 5micrograms/kg 1,25-(OH)₂ D₃.

7. One diet contained both 75 units of phytase plus 5 micrograms/kg1,25-(OH)₂ D₃.

8. One diet contained 0.2% supplemental phosphorus plus 75 units ofphytase plus 5 micrograms/kg 1,25-(OH)₂ D₃.

All chickens were fed a corn-soybean diet containing only 0.6% calcium,0.34% phosphorus, and a vitamin mixture supplying 1100 ICU D₃ /kg ofdiet.

The results are shown in Table 4 below.

ANALYSIS OF EXPERIMENT 2 RESULTS

The addition of 1,25-(OH)₂ D₃ (2) to the diet caused an increase in theutilization of phytate phosphorus by the broiler chickens from 31% to68%. The combination of phytase and 1,25-(OH)₂ D₃ raised the utilizationto 79%.

                                      TABLE 4                                     __________________________________________________________________________    Effect of 1,25-(OH).sub.2 D.sub.3 plus phytase                                                                               Retention                                                                                Metabo-                               Tibial dyschondroplasia                                                                          Blood plasma                                                                            the broiler                                                                              lizable                   Wt.     Bone         #3  Phos.     Dialyzable  Phytate                                                                            energy                    9-d Gain:                                                                             ash Incidence                                                                              scores                                                                            rickets                                                                             Ca  P     Ca P  P    of diet             Treatments                                                                          g   feed                                                                              %   %    Score                                                                             %   incidence                                                                           mg/kg                                                                             mg/kg %  %  %    kcal/kg             __________________________________________________________________________    Basal 0% P                                                                          145 .732                                                                              24.2                                                                               7   0.67                                                                              0   55    133.5                                                                             12.2  29.8                                                                             43.6                                                                             31.3 2.88                .2% P 170 .721                                                                              34.8                                                                              10   1.00                                                                              3    0    112.1                                                                             50.3  51.5                                                                             49.5                                                                             42.3 2.79                75 units                                                                            154 .748                                                                              23.7                                                                               7   0.67                                                                              0   60    126.0                                                                             11.9  30.6                                                                             44.1                                                                             41.6 2.98                phytase                                                                       5 μg/kg                                                                          172 .760                                                                              29.8                                                                              17   1.00                                                                              0   10    136.5                                                                             14.5  44.8                                                                             64.8                                                                             68.4 2.99                1,25-                                                                         (OH).sub.2 D.sub.3                                                            .2% P +                                                                             181 .772                                                                              35.4                                                                               7   1.33                                                                              3    0    118.3                                                                             61.3  49.3                                                                             51.6                                                                             44.4 2.82                75 units                                                                      phytase                                                                       .2% P +                                                                             186 .776                                                                              35.9                                                                               3   1.00                                                                              3    0    116.2                                                                             61.7  57.3                                                                             52.8                                                                             62.5 2.85                5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            75 units                                                                            167 .735                                                                              30.1                                                                              17   1.33                                                                              3   27    119.6                                                                             39.7  46.2                                                                             59.5                                                                             79.4 2.84                Phytase +                                                                     5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            .2% P +                                                                             178 .772                                                                              35.8                                                                              10   1.67                                                                              3    7    128.5                                                                             66.2  53.0                                                                             53.7                                                                             67.7 2.87                75 units                                                                      phytase                                                                       5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            Mean ±                                                                           169 ±                                                                          .752 ±                                                                         31.2 ±                                                                          10 ±                                                                           1.08 ±                                                                          2.08 ±                                                                        20 ±                                                                             123.8 ±                                                                        39.7 ±                                                                           45.3 ±                                                                        52.1 ±                                                                        54.7                                                                               2.88 ±           SEM    4  .016                                                                              .6  .06  .60 2.64                                                                              6     6.1 8.7   3.4                                                                              2.4                                                                              4.5  .03                 __________________________________________________________________________

EXPERIMENT 3

Each of the twelve different dietary treatments described below wereprovided to two pens containing ten birds per pen.

1. One diet was a basal diet lacking supplemental phytase or 1,25-(OH)₂D₃ (2).

2. Five diets contained graded levels of phytase from 37.5 units to 600units.

3. One diet contained basal phytase plus 5 microgram/kg feed of1,25-(OH)₂ D₃.

4. Five diets contained graded levels of phytase plus 5 microgram/kgfeed of 1,25-(OH)₂ D₃.

The results are shown in Table 5 below. In Table 5, and subsequenttables, the abbreviations TDI and TDS represent tibial dyschondroplasiaincidence and tibial dyschondroplasia score, respectively. N3 indicatesthe number 3 scores for tibial dyschondroplasia. Ri is an abbreviationfor rickets incidence. B.A. is an abbreviation for bone ash. CAR, PR,and PPR represent calcium retention, phosphorus retention and phytatephosphorus retention respectively.

ANALYSIS OF EXPERIMENT 3 RESULTS

The addition of 1,25-(OH)₂ D₃ (2) to the diet caused an increase theutilization of phytate phosphorus by the broiler chickens which wasapparent from growth, bone ash, blood calcium, and total phosphorusretention. The addition of 1,25-(OH)₂ D₃ in the presence of gradedlevels of phytase resulted in a gradual increase in the phytatephosphorus utilization from 67% when no phytase was present to 89% when600 units of phytase/kg of feed was present in the diet.

                                      TABLE 5                                     __________________________________________________________________________    Effect of 1,25-(OH).sub.2 D.sub.3 plus graded levels of phytase                         9-d Tibial dyschondroplasia                                                                           Blood plasma                                          wt. TDI   N3 Ri     B.A.                                                                              Ca  Dialyzable P                                                                         Survivors                                                                           CAR     PPR                Treatments                                                                              g   %  TDS                                                                              %  %  G:F %   mg/kg                                                                             mg/kg  %     %   PR  %                  __________________________________________________________________________    Basal     147 20 2.00                                                                             6  62 .692                                                                              23.7                                                                              9.83                                                                              4.70   16/20 30.87                                                                             41.58                                                                             48.63               37.5 units Phytase                                                                     172 19 1.00                                                                             0  32 .724                                                                              26.6                                                                              11.83                                                                             2.50   16/20 36.28                                                                             43.61                                                                             66.38               75 units Phytase                                                                       161  0  .00                                                                             0  60 .715                                                                              23.3                                                                              12.10                                                                             1.70   20/20 26.60                                                                             37.88                                                                             49.68              150 units Phytase                                                                       182 10  .75                                                                             0  70 .768                                                                              24.0                                                                              13.57                                                                             .75    20/20 17.63                                                                             33.92                                                                             52.01              300 units Phytase                                                                       182  5  .50                                                                             0  60 .789                                                                              24.3                                                                              12.63                                                                             1.20   20/20 17.83                                                                             42.39                                                                             54.15              600 units Phytase                                                                       185  0  .00                                                                             0  30 .764                                                                              26.4                                                                              13.38                                                                             .85    20/20 19.83                                                                             47.08                                                                             73.31              Basal + 5 μg                                                                         187 10  .50                                                                             0  40 .808                                                                              26.9                                                                              14.30                                                                             1.15   18/20 31.14                                                                             55.89                                                                             66.59              1,25-(OH).sub.2 D.sub.3                                                       37.5 Phytase + 5 μg                                                                  184 15 2.00                                                                             5  25 .773                                                                              29.9                                                                              14.92                                                                             .95    20/20 37.28                                                                             57.03                                                                             79.11              1,25-(OH).sub.2 D.sub.3                                                       75 Phytase + 5 μg                                                                    181  0  .00                                                                             0  25 .776                                                                              28.7                                                                              13.60                                                                             1.70   20/20 35.73                                                                             57.74                                                                             80.07              1,25-(OH).sub.2 D.sub.3                                                       150 Phytase + 5 μg                                                                   189  6  .50                                                                             0  36 .752                                                                              28.4                                                                              14.43                                                                             .95    19/20 34.91                                                                             56.90                                                                             84.09              1,25-(OH).sub.2 D.sub.3                                                       300 Phytase + 5 μg                                                                   194 25 2.00                                                                             5  20 .788                                                                              30.1                                                                              12.99                                                                             .78    20/20 42.87                                                                             61.41                                                                             84.89              1,25-(OH).sub.2 D.sub.3                                                       600 Phytase + 5 μg                                                                   187 21 1.00                                                                             0  11 .766                                                                              32.5                                                                              14.50                                                                             1.75   19/20 44.09                                                                             61.25                                                                             88.74              1,25-(OH).sub.2 D.sub.3                                                       Mean ± SEM                                                                           179 ±                                                                          11 ±                                                                           .85 ±                                                                        1 ±                                                                           39 ±                                                                          .760 ±                                                                         27.1 ±                                                                         13.2 ±                                                                         1.58 ±    31.25                                                                             49.71                                                                             68.97 ±                    4   7  .60                                                                             3  10 .015                                                                              .7  1.0 .64          4.89                                                                              3.77                                                                              7.90               __________________________________________________________________________

EXPERIMENT 4

The objective of experiment 4 was to determine if the addition of1,25-(OH)₂ D₃ to a corn-soybean diet containing 0.6% calcium wouldenhance the utilization of phytate phosphorus in the presence of variouslevels of phosphate.

Each of the twelve different dietary treatments set forth below wereprovided to three pens containing ten birds per pen.

1. One diet was a basal diet lacking supplemental inorganic phosphorus,phytase or 1,25-(OH)₂ D₃ (2).

2. Two diets contained graded levels of phosphorus (0.1and 0.2%) fromreagent grade Na₂ HPO₄ H₂ O.

3. One diet contained basal phosphate plus 150 units of phytase.

4. Two diets contained graded levels of phosphorus (0.1 and 0.2%) plus150 units of phytase (Sigma Chemical Co., St. Louis, Mo.).

5. One diet contained basal levels of phosphorus plus 5 microgram/kgfeed of 1,25-(OH)₂ D₃.

6. Two diets contained graded levels of phosphorus (0.1 and 0.2%) plus 5microgram/kg feed of 1,25-(OH)₂ D₃.

7. One diet contained basal levels of phosphorus plus phytase plus 5micrograms/kg 1,25-(OH)₂ D₃.

8. Two diets contained graded levels of phosphorus (0.1 and 0.2%) plus150 units of phytase plus 5 microgram/kg feed of 1,25-(OH)₂ D₃.

All chickens were fed a corn-soybean diet containing only 0.6% calcium,0.34% phosphorus, and a vitamin mixture supplying 1100 ICU D₃ /kg ofdiet.

The results are shown in Table 6 below.

ANALYSIS OF EXPERIMENT 4 RESULTS

The addition of inorganic phosphate to the diet decreases phytatephosphorus utilization and decreases the effectiveness of 1,25-(OH)₂ D₃(2) in increasing phytate phosphorus utilization.

                                      TABLE 6                                     __________________________________________________________________________    Effects of phosphorus, phytase and 1,25-(OH).sub.2 D.sub.3                         9-d wt.                                                                           TDI    N3 Ri     Ri#3   B.A.                                                                              TCa DP                                   Diets                                                                              g   %  TDS %  %  RiS %  G:F %   %   %   Survivors                                                                           CAR PR  PPR                __________________________________________________________________________    Basal                                                                              153 13 1.00                                                                               6 75 3.00                                                                               7 .693                                                                              24.3                                                                              12.33                                                                             1.22                                                                              18/20 39.02                                                                             49.04                                                                             53.89              .1% P                                                                              187  0 0.00                                                                               0 50 3.00                                                                              50 .769                                                                              31.8                                                                              11.95                                                                             1.53                                                                              20/20 30.08                                                                             53.18                                                                             53.63              .2% P                                                                              202 22 3.00                                                                              22 35 3.00                                                                              38 .784                                                                              34.2                                                                              10.60                                                                             4.22                                                                              19/20 53.77                                                                             54.25                                                                             48.02              150 units                                                                          188 10 1.00                                                                               5 90 3.00                                                                              90 .801                                                                              24.7                                                                              13.37                                                                             .70 20/20 35.09                                                                             50.07                                                                             63.49              Phytase                                                                       conc.                                                                         .1% P +                                                                            193 25 1.30                                                                              20 30 3.00                                                                              30 .771                                                                              32.1                                                                              10.29                                                                             2.74                                                                              20/20 45.17.sup.abc                                                                     57.48                                                                             58.86              150 units                                                                     Phytase                                                                       conc.                                                                         .2% P +                                                                            203 11 1.50                                                                              11  6 1.50                                                                               6 .804                                                                              34.8                                                                              11.13                                                                             4.37                                                                              19/20 53.10.sup.                                                                        53.23                                                                             51.36              150 units                                                                     Phytase                                                                       conc.                                                                         Basal +                                                                            187  0 0.00                                                                               0 40 3.00                                                                              40 .732                                                                              30.8                                                                              13.88                                                                             1.19                                                                              20/20 48.93.sup.abc                                                                     65.89                                                                             79.40              5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            .1% P +                                                                            193  5 1.50                                                                               5 10 3.00                                                                              10 .795                                                                              34.3                                                                              13.23                                                                             3.02                                                                              20/20 51.16                                                                             62.64                                                                             72.16              5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            .2% P +                                                                            198 16 3.00                                                                              16 15 3.00                                                                              15 .817                                                                              35.8                                                                              11.33                                                                             4.73                                                                              19/20 55.69                                                                             57.72                                                                             72.66              5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            Basal +                                                                            181 10 3.00                                                                              10 25 3.00                                                                              25 .743                                                                              31.6                                                                              11.98                                                                             2.43                                                                              20/20 50.17                                                                             66.11                                                                             76.64              150 units                                                                     Phytase +                                                                     5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            .1% P +                                                                            199 10 2.00                                                                               5  5 1.50                                                                               5 .810                                                                              35.6                                                                              11.68                                                                             3.36                                                                              20/20 56.02                                                                             62.63                                                                             75.02              150 units                                                                     Phytase +                                                                     5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub.3                                                            .2% P +                                                                            182  0 0.00                                                                               0  5 1.50                                                                               5 .709                                                                              36.1                                                                              10.65                                                                             4.54                                                                              20/20 54.44                                                                             49.08                                                                             63.85              150 units                                                                     Phytase +                                                                     5 μg/kg                                                                    1,25-                                                                         (OH).sub.2 D.sub. 3                                                           Mean ±                                                                          188 ±                                                                          10 ±                                                                          1.44 ±                                                                          8 ±                                                                          32 ±                                                                          2.63 ±                                                                         32 ±                                                                          .769 ±                                                                         32.2 ±                                                                         11.87 ±                                                                        2.83 ± 47.72                                                                             56.77                                                                             64.08 ±         SEM   6  10  .88                                                                               8 16  .75                                                                              17 .020                                                                              .8  .06 .45        5.90                                                                             2.63                                                                              7.176              __________________________________________________________________________

EXPERIMENT 5

The objective of experiment 5 was to determine if the addition

1,25-(OH)₂ D₃ (2) to a corn-soybean meal diet with three levels ofcalcium with and without added phytase would enhance the utilization ofphytate phosphorus.

Each of the twelve different dietary treatments set forth below wereprovided to three pens containing ten birds per pen.

1. Three diets contained graded levels of calcium (0.40%, 0.57% and0.72%). The concentration of 0.40% calcium was obtained by adding 17.6grams limestone per kilograms feed. The concentration of 0.57% calciumwas obtained by adding 35.2 g/kg limestone. The 0.72% calcium value wasobtained by adding 52.8 g/kg limestone.

2. Three diets contained 75 units phytase per kilogram feed plus each ofthe graded levels of calcium.

3. Three diets contained 5 microgram/kg feed of 1,25-(OH)₂ D₃ plus eachof the graded levels of calcium.

4. Three diets contained 5 microgram/kg feed of 1,25-(OH)₂ D₃ plusphytase plus the three graded levels of calcium.

All chickens were fed a corn-soybean diet containing the diet specifiedabove and a vitamin mixture supplying 1100 ICU D₃ /kg of diet.

The results are shown in Table 7 below.

ANALYSIS OF EXPERIMENT 5 RESULTS

Feeding the low calcium diet (0.40%) made it possible to obtain a veryhigh level of phytate phosphorus retention (86%) with the basal dietcontaining no added phytase or 1,25-(OH)₂ D₃ (2) to the diet. Theaddition of phytase or 1,25-(OH)₂ D₃ to the diet resulted in slightlygreater phytate utilization. However, greater phytate utilization wasapparent when both phytase and 1,25-(OH)₂ D₃ were added to the diet,especially in the higher dietary calcium levels.

                                      TABLE 7                                     __________________________________________________________________________    Effects of 1,25-(OH).sub.2 D.sub.3 and graded levels of calcium               Treat-                                                                             9-d wt.                                                                           TDI    N3 Ri     Ri#3   B.A.                                                                              TCa DP        CAR PR  PPR                ments                                                                              g   %  TDS %  %  RiS %  G:F %   %   %   Survivors                                                                           %   %   %                  __________________________________________________________________________    0.40%                                                                              177  5 1.50                                                                               5 90 3.00                                                                              90 .765                                                                              28.1                                                                              8.18                                                                              7.90                                                                              19/20 68.61                                                                             37.66                                                                             86.21              Calcium                                                                       0.57%                                                                              207 15 1.50                                                                               5 90 2.11                                                                              50 .806                                                                              32.8                                                                              9.43                                                                              8.15                                                                              19/20 60.57                                                                             49.07                                                                             78.39              Calcium                                                                       0.72%                                                                              192 15 1.25                                                                               5 65 1.91                                                                              30 .780                                                                              34.2                                                                              10.60                                                                             4.70                                                                              20/20 51.51                                                                             52.01                                                                             55.16              Calcium                                                                       0.40%                                                                              189  5 1.50                                                                               5 95 2.80                                                                              85 .825                                                                              28.5                                                                              8.63                                                                              9.30                                                                              20/20 62.27                                                                             46.34                                                                             89.37              Calcium +                                                                     Phytase                                                                       0.57%                                                                              201 15 3.00                                                                              15 60 2.12                                                                              35 .827                                                                              33.3                                                                              9.88                                                                              7.10                                                                              20/20 58.79                                                                             52.56                                                                             69.95              Calcium +                                                                     Phytase                                                                       0.72%                                                                              200 37 2.42                                                                              26 64 2.75                                                                              53 .797                                                                              35.7                                                                              11.25                                                                             6.95                                                                              19/20 52.40                                                                             54.84                                                                             63.38              Calcium +                                                                     Phytase                                                                       0.40%                                                                              185 15 1.50                                                                               5 75 2.45                                                                              55 .732                                                                              30.9                                                                              9.33                                                                              7.45                                                                              20/20 64.53                                                                             39.67                                                                             94.42              Calcium +                                                                     1,25-                                                                         (OH).sub.2 D.sub.3                                                            0.57%                                                                              211 21 2.25                                                                              16 70 2.40                                                                              42 .831                                                                              34.8                                                                              10.58                                                                             7.30                                                                              19/20 64.07                                                                             53.27                                                                             78.67              Calcium +                                                                     1,25-                                                                         (OH).sub.2 D.sub.3                                                            1.89%                                                                              160  0 0.00                                                                               0 16 1.50                                                                               6 .759                                                                              34.1                                                                              13.30                                                                             4.60                                                                              19/20 50.40                                                                             47.46                                                                             37.84              Calcium.sup.1 +                                                               1,25-                                                                         (OH).sub.2 D.sub.3                                                            0.40%                                                                              190 20 2.50                                                                              15 85 2.75                                                                              75 .764                                                                              31.1                                                                              9.73                                                                              7.70                                                                              20/20 71.88                                                                             40.91                                                                             90.87              Calcium +                                                                     1,25-                                                                         (OH).sub.2 D.sub.3 +                                                          Phytase                                                                       0.57%                                                                              185 15 0.50                                                                               0 70 1.25                                                                              10 .716                                                                              35.3                                                                              10.35                                                                             8.25                                                                              20/20 59.73                                                                             44.49                                                                             96.70              Calcium +                                                                     1,25-                                                                         (OH).sub.2 D.sub.3 +                                                          Phytase                                                                       0.72%                                                                              191 22 2.00                                                                              11 28 1.84                                                                              11 .786                                                                              36.7                                                                              11.10                                                                             7.90                                                                              20/20 63.00                                                                             56.63                                                                             75.51              Calcium +                                                                     1,25-                                                                         (OH).sub.2 D.sub.3 +                                                          Phytase                                                                       Mean ±                                                                          191 ±                                                                          15 ±                                                                          1.66 ±                                                                          9 ±                                                                          67 ±                                                                          2.24 ±                                                                         45 ±                                                                          .782 ±                                                                         33.0 ±                                                                         10.20                                                                             7.28 ± 60.64                                                                             48.07                                                                             76.20 ±         SEM   7   6  .72                                                                               4  9  .26                                                                               9 .029                                                                              .6  .71 .80       2.52                                                                              3.90                                                                              2.74               __________________________________________________________________________     .sup.1 There was a mixing error in preparing the diet that should have        contained 0.72% calcium and 1,25(OH).sub.2 D.sub.3. The diet mixed and fe     actually contained 1.89% calcium.                                        

EXPERIMENT 6

The objective of experiment 6 was to determine if the addition of thevitamin D₃ derivatives 1,25-(OH)₂ D₃ (2), 1-OHD₃ (4) or 24,25-(OH)₂ D₃(8) to a corn-soybean meal diet with and without added phytase wouldenhance the utilization of phytate phosphorus.

Each of the eight different dietary treatments set forth below wereprovided to two pens containing ten birds per pen.

1. One diet contained basal levels of phytase.

2. One diet contained 150 units of phytase.

3. Three diets contained 5 micrograms/kg of either 1,25-(OH)₂ D₃ (2),1-OHD₃ (4), or 24,25-(OH)₂ D₃ (8).

4. Three diets contained 150 units of phytase plus 5 micrograms/kg ofeither 1,25-(OH)₂ D₃ (2), 1-OHD₃ (4), or 24,25-(OH)₂ D₃ (8).

All chickens were fed a corn-soybean diet containing the diet specifiedabove and a vitamin mixture supplying 1100 ICU D₃ /kg of diet.

The results are shown in Table 8 below.

ANALYSIS OF EXPERIMENT 6 RESULTS

The 1,25-(OH)₂ D₃ (2) and 1-OHD₃ (4) supplementation of the dietresulted in increased nine-day weights and bone ash as contrasted tosupplementation with 24,25-(OH)₂ D₃ (8) which had no effect on thesecriteria in the presence or absence of supplemental phytase. In thisstudy, the 1-OHD₃ (4) and 24,25-(OH)₂ D₃ (8) did not appear to increasephytate phosphorus utilization in the absence of phytase.Supplementation with 1,25-(OH)₂ D₃ (2) increased phytate phosphorusutilization in the presence or absence of phytase.

                                      TABLE 8                                     __________________________________________________________________________    Effects of 1,25-(OH).sub.2 D.sub.3, 1-OHD.sub.3, and 24,25-(OH).sub.2         D.sub.3                                                                                  9-d           Ri                                                              wt. TDI    TD I  Ri  Ri     B.A.                                                                              TCa DP                             Treatments g   %  TDS N3 %  S   #3 G:F %   %   %   CAR PR  PPR                __________________________________________________________________________    Basal      143  7 1.33                                                                              3  86 3.00                                                                              86 .655                                                                              24.3                                                                              11.92                                                                             1.63                                                                              40.29                                                                             49.18                                                                             61.11              150 Phytase                                                                              159  8 1.33                                                                              4  82 3.00                                                                              79 .685                                                                              24.9                                                                              13.25                                                                             .54 37.49                                                                             47.47                                                                             57.18              150 Phytase +                                                                            166 17 1.11                                                                              7  53 3.00                                                                              53 .704                                                                              29.6                                                                              15.63                                                                             1.27                                                                              40.53                                                                             62.28                                                                             80.29              1,25-(OH).sub.2 D.sub.3                                                       150 Phytase +                                                                            168 17 2.11                                                                              7  58 3.00                                                                              59 .741                                                                              28.1                                                                              14.47                                                                             .67 41.76                                                                             53.61                                                                             69.37              1-OHD.sub.3                                                                   150 Phytase +                                                                            152  7  .67                                                                              4  75 3.00                                                                              82 .716                                                                              24.3                                                                              11.77                                                                             2.07                                                                              33.32                                                                             47.05                                                                             69.17              24,25-(OH).sub.2 D.sub.3                                                      1,25-(OH).sub.2 D.sub.3                                                                  168  4 1.00                                                                              4  24 3.00                                                                              25 .705                                                                              31.7                                                                              12.50                                                                             1.93                                                                              54.57                                                                             65.27                                                                             72.93              1-OHD.sub.3                                                                              160 10  .67                                                                              0  77 3.00                                                                              77 .718                                                                              26.1                                                                              14.77                                                                             .86 37.15                                                                             49.67                                                                             61.29              24,25-(OH).sub.2 D.sub.3                                                                 150  4 1.00                                                                              4  72 3.00                                                                              71 .707                                                                              23.5                                                                              14.84                                                                             .61 30.99                                                                             49.65                                                                             62.45              Mean ± SEM                                                                            158 ±                                                                           9 ±                                                                          1.11 ±                                                                         4 ±                                                                           66 ±                                                                          3.00 ±                                                                         66 ±                                                                          .704 ±                                                                         26.6 ±                                                                         13.61 ±                                                                        1.20 ±                                                                         39.57                                                                             53.21                                                                             67.07 ±                     4   6  .80                                                                              4   8 0    8 .023                                                                              .4  .89 .39 2.44                                                                              2.41                                                                              7.74               __________________________________________________________________________

EXPERIMENT 7

The objective of experiment 7 was to determine if the addition thevitamin D₃ derivatives 1,24,25-(OH)₃ D₃ (5), 1,25-(OH)₂ -24-FD₃ (6), or1,25-(OH)₂ -26,27[³ H]₆ D₃ (3) to a corn-soybean meal diet with andwithout added phytase would enhance the utilization of phytatephosphorus.

Each of the eight different dietary treatments set forth below wereprovided to two pens containing ten birds per pen.

1. One diet contained basal levels of phytase and vitamin D₃derivatives.

2. One diet contained 150 units of phytase.

3. Three diets contained 5 micrograms/kg of either 1,24,25-(OH)₃ D₃ (5),1,25-(OH)₂ -24-FD₃ (6) or 1,25-(OH)₂ -26,27[³ H]₆ D₃ (3).

4. Three diets contained 150 units of phytase plus 5 micrograms/kg ofeither 1,24,25-(OH)₃ D₃ (5), 1,25-(OH)₂ -24-FD₃ (6) or 1,25-(OH)₂-26,27[³ H]₆ D₃ (3).

All chickens were fed a corn-soybean diet containing the diet specifiedabove and a vitamin mixture supplying 1100 ICU D₃ /kg of diet.

The results are shown in Table 9 below.

ANALYSIS OF EXPERIMENT 7 RESULTS

All three of the D₃ derivatives increased the utilization of phytatephosphorus by the broiler chickens, but 1,25-(OH)₂ -26,27 [³ H]₆ D₃ (3)was more effective than the other two vitamin D₃ derivatives.

                                      TABLE 9                                     __________________________________________________________________________    Effects of 1,24,25-(OH).sub.3 D.sub.3,                                        1,25-(OH).sub.2 -24-FD.sub.3, and 1,25-(OH).sub.2 -26,27[.sup.3 H].sub.6      D.sub.3                                                                                  9-d           Ri                                                              wt. TDI    TD I  Ri  Ri     B.A.                                                                              TCa DP                             Treatments g   %  TDS N3 %  S   #3 G:F %   %   %   CAR PR  PPR                __________________________________________________________________________    Basal      155 10 1.00                                                                              3  53 2.90                                                                              50 .703                                                                              25.79                                                                             10.38                                                                             1.37                                                                              40.12                                                                             46.32                                                                             53.11              150 units Phytase                                                                        158 10 1.00                                                                              0  73 3.00                                                                              73 .728                                                                              24.19                                                                             10.92                                                                             .31 39.73                                                                             53.96                                                                             63.61              Phytase +  168 7  .66 0  90 2.92                                                                              87 .750                                                                              25.78                                                                             11.18                                                                             .21 43.65                                                                             55.93                                                                             64.53              1,24,25-(OH).sub.3 D.sub.3                                                    Phytase +  161 18 1.00                                                                              0  65 2.89                                                                              61 .700                                                                              27.04                                                                             10.67                                                                             .64 44.66                                                                             56.98                                                                             66.85              1,25-(OH).sub.2 -                                                             24R-FD.sub.3                                                                  Phytase +  169 7  .33 0  37 3.00                                                                              37 .749                                                                              31.00                                                                             10.55                                                                             2.40                                                                              50.65                                                                             70.09                                                                             81.47              1,25-(OH).sub.2 -                                                             26,27[.sup.3 H].sub.6 D.sub.3                                                 1,24,25-(OH).sub.3 D.sub.3                                                               151 7  .33 0  79 2.93                                                                              76 .680                                                                              26.27                                                                             10.58                                                                             .44 46.90                                                                             54.68                                                                             67.71              1,25-(OH).sub.2 -                                                                        162 18 1.66                                                                              4  72 3.00                                                                              72 .737                                                                              27.78                                                                             10.68                                                                             .74 47.36                                                                             53.39                                                                             63.12              24R-FD.sub.3                                                                  1,25-(OH).sub.2 -                                                                        160 3  .33 0  30 3.00                                                                              30 .716                                                                              30.25                                                                             10.12                                                                             1.57                                                                              48.87                                                                             62.08                                                                             77.03              26,27[.sup.3 H].sub.6 D.sub.3                                                 Mean ± SEM                                                                            160 ±                                                                          10 ±                                                                          .83 ±                                                                          1 ±                                                                           63 ±                                                                          2.95 ±                                                                         61 ±                                                                          .720 ±                                                                         27.26 ±                                                                        10.64 ±                                                                        .96 ±                                                                          45.24                                                                             56.68                                                                             67.18 ±                     5    .6                                                                             .41 2   9 .07  9 .017                                                                              .64 .64 .40 3.54                                                                              2.30                                                                              4.19               __________________________________________________________________________

EXAMPLE 2: TREATMENT OF TIBIAL DYSCHONDROPLASIA WITH VITAMIN D₃DERIVATIVES

The effectiveness of 1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5), and1,25-dihydroxy-24-fluorocholecalciferol (6) in reducing the incidence oftibial dyschondroplasia in chickens has been demonstrated using thefollowing experimental procedures.

Newly hatched Peterson X Arbor Acre cockerels obtained from a commercialhatchery were housed as described in Example 1 above. The basal dietused is described above in Table 2. Disulfiram was added to the basaldiet of some chickens as indicated in Table 10 at a level of 30 mg/kg offeed. The following levels of vitamin D₃ and its metabolites were addedto the diet as indicated in Tables 10 and 11:

Vitamin D₃, 27.5 micrograms per kilogram of feed (1100 ICU/kilogram);and

1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24(R),25-trihydroxycholecalciferol (5),1,25-dihydroxy-24(R)-fluorocholecalciferol (6), and25-hydroxycholecalciferol (7) at 10 micrograms per kilogram.

At 16 days of age, pen body weights were obtained for all birds andtheir feed consumption was recorded. The birds were then sacrificed andexamined at random for tibial dyschondroplasia by making a longitudinalcut across the tibia and scoring for incidence and severity of tibialdyschondroplasia as described in Example 1 above. After the tibia wasscored for incidence and severity of tibial dyschondroplasia, the lefttibia was removed for a bone ash determination on the fat-free driedbone.

Absorption and retention of dietary calcium was measured by orallydosing chicks with ⁴⁷ Ca and then measuring the total body radioactivecount, as described in Suso and Edwards, "A Study of Techniques forMeasuring ⁶⁵ Zn Absorption and Biological Half-life in the Chicken,"Poult. Sci. 47:991-999 (1968). Specifically, ten seven day old birds inone pen under the indicated dietary treatments were dosed orally with0.5 milliliter of water solution containing 0.5 microcuries of ⁴⁷ Ca and0.1 to 1.0 micrograms of calcium. The total body radioactive content ofthe individual chickens was determined immediately following dosing, andalso on days 7, 9, 12 and 14 following dosing. A plot of administered ⁴⁷Ca which was retained versus number of days yields a line which has aslope corresponding to the half-life of the radioactive calcium in thechicken, and a y-intercept that corresponds to the amount of calciumabsorbed by the chicken.

EFFECT OF VITAMIN D₃ DERIVATIVES ON TIBIAL DYSCHONDROPLASIA

The effect in broiler cockerels of vitamin D₃ (1),25-hydroxycholecalciferol (7), and 1,25-dihydroxycholecalciferol (2) ongrowth, feed efficiency, bone ash, ⁴⁷ Ca retention, and incidence andseverity of tibial dyschondroplasia was determined in Studies 1 and 2.The results are shown in Table 10 below. The basal diet contained 1100ICU (27.5 micrograms) vitamin D₃ per kilogram feed. Disulfiram was addedas indicated to the diets of some chickens to increase the incidence ofthe disease in order to further measure the effectiveness of thecompounds.

As shown below in Table 10, 1,25-dihydroxycholecalciferol (2), in thepresence or absence of disulfiram, reduced the incidence and severity oftibial dyschondroplasia over the basal diet, and increased bone ash.25-(OH)-Cholecalciferol (7), in the presence or absence of disulfiram,also reduced the incidence of tibial dyschondroplasia over the basaldiet. Further, a diet with 1,25-dihydroxycholecalciferol resulted insignificantly more dietary ⁴⁷ Ca absorbed than in a basal diet, whileincreasing the half-life, or retention, of the calcium administered.

Table 11, shown below, illustrates the results of Studies 3 and 4, inwhich the effect of vitamin D₃ (cholecalciferol) (1),1,25-dihydroxycholecalciferol (2),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3),1-hydroxycholecalciferol (4), 1,24,25-trihydroxycholecalciferol (5),1,25-dihydroxy-24-fluorocholecalciferol (6), and24,25-dihydroxycholecalciferol (8), on the incidence and severity oftibial dyschondroplasia, bone ash and absorption and retention ofcalcium was measured. The basal diet contained 1100 ICU of vitamin D₃per kilogram feed.

In Tables 10 and 11, values with different superscript letters aresignificantly different, P≦0.05.

Studies 3 and 4 substantiated the results of studies 1 and 2 thatadministration of 10 microgram/kg feed of 1,25-dihydroxycholecalciferol(2) to the bird per day for the life of the bird substantially reducesthe incidence and severity of tibial dyschondroplasia over the basaldiet, and increases bone ash.

The addition of 24,25-dihydroxycholecalciferol (8) was not effective inpreventing the development of tibial dyschondroplasia.

The chickens that received feed containing1,24,25-trihydroxycholecalciferol (5), 1-hydroxycholecalciferol (4),1,25-dihydroxy-26,27-hexadeuterocholecalciferol (3) and1,25-dihydroxy-24-fluorocholecalciferol (6) had a significant reductionin the incidence of tibial dyschondroplasia.

                                      TABLE 10                                    __________________________________________________________________________    Effect of Vitamin D.sub.3 ; 25-OHD.sub.3 and 1,25-(OH).sub.2 D.sub.3 on       Severity of Tibial Dyschondroplasia                                           With and Without Disulfiram                                                   __________________________________________________________________________    Study 1                                                                                     16 Day         Bone  Tibial Dyschondroplasia                    Treatments    wt     Gain:Feed                                                                             ash   Incidence                                                                            Score #3/total                      __________________________________________________________________________    Basal         416    .696    40.6  42     2.77  10/30                         Basal + D.sub.3                                                                             451    .734    40.4  26     2.38  4/27                          Basal + 25-OHD.sub.3                                                                        395    .667    40.2  17     1.50  1/28                          Basal + 1,25-(OH).sub.2 D.sub.3                                                             421    .670    42.5  13     1.75  1/29                          Disulfiram    433    .750    39.9  70     2.71  16/30                         Disulfiram + D.sub.3                                                                        432    .708    40.0  40     2.80  9/27                          Disulfiram + 25-OHD.sub.3                                                                   398    .706    39.7  58     2.54  11/30                         Disulfiram + 1,25-(OH).sub.2 D.sub.3                                                        426    .701    41.9  23     2.33  4/30                          Means ± SEM                                                                              420 ± 13                                                                          .704    .032  40.6 ± .2                                                                         36 ± 9                                                                           2.11 ± .48                 Study 2                                                                                     16 day    Bone Tibial Dyschondroplasia                                                                      .sup.47 Ca                        Treatments    wt  Gain:Feed                                                                           ash  Incidence                                                                          Score                                                                             #3/total                                                                            Intercept                                                                          Slope                        __________________________________________________________________________    Basal         375 .707  40.5 39   1.94                                                                              6/29  72   -1.60                        Basal + D.sub.3                                                                             368 .647  40.5 31   2.47                                                                              5/29  77   -1.88                        Basal + 25OHD.sub.3                                                                         392 .713  40.6 27   2.78                                                                              7/30  72   -1.59                        Basal + 1,25(OH).sub.2 D.sub.3                                                              361 .661  42.2 13   2.17                                                                              2/30  84   -2.85                        Disulfiram    394 .671  40.3 65   2.76                                                                              14/28 73   -1.59                        Disulfiram + D.sub.3                                                                        374 .666  40.3 51   2.74                                                                              12/30 72   -1.97                        Disulfiram + 25OHD.sub.3                                                                    395 .728  40.5 49   2.76                                                                              11/28 74   -1.67                        Disulfiram + 1,25(OH).sub.2 D.sub.3                                                         377 .684  42.0 30   2.44                                                                              6/30  79   -2.11                        Means ± SEM                                                                              380 ±                                                                          .685 ±                                                                           40.8 ±                                                                           38 ±                                                                            2.51 ±                                                                               75 ±                                                                           -1.91 ±                                 12  .026   .3   9    .31       3   .29                          __________________________________________________________________________

                                      TABLE 11                                    __________________________________________________________________________    Effect of Vitamin D3 and Vitamin D3 Derivatives on Severity of Tibial         Dyschondroplasia                                                              __________________________________________________________________________    Study 3                                                                                16 Day                                                                             Gain/ Tibial Dyschondroplasia   Ca.sup.47                       Vit D Compound                                                                         Wt.  Feed  Incidence                                                                          Score                                                                             #3/No.                                                                              % N3 % N2 + 3                                                                            Ret.                                                                              Slope                       __________________________________________________________________________    none     421  .711.sup.a                                                                          63   1.85                                                                              11/60 18   37    70.sup.bc                                                                         -1.01.sup.b                 D.sub.3  434  .709.sup.a                                                                          82   1.69                                                                              13/59 22   34    75.sup.abc                                                                        -1.13.sup.b                 1,25(OH).sub.2 D.sub.3                                                                 421  .66.sup.b                                                                           56   1.48                                                                              5/58   9   16    78.sup.ab                                                                         -.72.sup.ab                 24,25(OH).sub.2 D.sub.3                                                                424  .696.sup.ab                                                                         78   2.03                                                                              17/59 29   53    67.sup.c                                                                          -.77.sup.ab                 1,24,25(OH).sub.2 D.sub.3                                                              428  .688.sup.ab                                                                         57   1.58                                                                              6/60  10   22    76.sup.abc                                                                        -.77.sup.ab                 1-OHD.sub.3                                                                            423  .661.sup.b                                                                          42   1.51                                                                              3/60   5   17    75.sup.abc                                                                        -.95.sup.b                  1,25(OH).sub.2 -26-                                                                    422  .687.sup.ab                                                                         42   1.30                                                                              0/60   0    8    80.sup.a                                                                          .15.sup.a                   27[.sup.2 H].sub.6 D.sub.3                                                    1,25(OH).sub.2 -                                                                       413  .663.sup.b                                                                          60   1.36                                                                              3/60    5  15    72.sup.abc                                                                        -.47.sup.ab                 24R-FD.sub.3                                                                  Mean ± SEM                                                                          423 ± 7                                                                         .685 ± 0.13                                                                           59 ± 7                                                                         1.60 ± 15                                                                        12 ± 4.5                                                                              74 ± 3                                                                         -.71 ± .31               __________________________________________________________________________    Study 4                                                                                   16 Day                                                                              Gain/ Bone  Tibial Dyschondroplasia                         Vit. D Compound                                                                           wt.   feed  ash   Incidence                                                                            Score % #3   #3/Total                    __________________________________________________________________________    None (basal diet)                                                                         356.sup.cdl                                                                         .681.sup.ab                                                                         35.07.sup.c                                                                         54.sup.a                                                                             2.30.sup.ab                                                                         35.sup.a                                                                             19/55                       D.sub.3     383.sup.a                                                                           .701.sup.a                                                                          35.68.sup.bc                                                                        44.sup.ab                                                                            2.75.sup.a                                                                          35.sup.a                                                                             20/58                       1,25(OH).sub.2 D.sub.3                                                                    355.sup.cd                                                                          .652.sup.b                                                                          36.88.sup.a                                                                         18.sup.d                                                                             1.58.sup.b                                                                           8.sup.b                                                                             4/52                        24,25(OH).sub.2 D.sub.3                                                                   351.sup.d                                                                           .663.sup.b                                                                          35.53.sup.bc                                                                        53.sup.a                                                                             2.78.sup.a                                                                          46.sup.a                                                                             25/56                       1,24,25(OH).sub.2 D.sub.3                                                                 376.sup.ab                                                                          .681.sup.ab                                                                         36.07.sup.b                                                                         23.sup.cd                                                                            1.89.sup.ab                                                                         14.sup.b                                                                             8/57                        1OHD.sub.3  372.sup.abc                                                                         .655.sup.b                                                                          36.77.sup.a                                                                         29.sup.bcd                                                                           2.21.sup.ab                                                                         15.sup.b                                                                             8/57                        1,25(OH).sub.2 -26-27[.sup.2 H].sub.6 D.sub.3                                             360.sup.bcd                                                                         .646.sup.b                                                                          37.45.sup.a                                                                         16.sup. d                                                                            1.63.sup.b                                                                           7.sup.b                                                                             4/58                        1,25(OH).sub.2 -24R-FD.sub.3                                                              360.sup.bcd                                                                         .661.sup.b                                                                          35.48.sup.bc                                                                        41.sup.abc                                                                           2.06.sup.ab                                                                         16.sup.b                                                                             9/25                        Mean ± SEM                                                                             364 ±                                                                            .667 ±                                                                           36.12 ±                                                                          34 ±                                                                              2.15 ±                                                                           22 ±                                         6    .012   .23   6      .31   5                                 __________________________________________________________________________

Modifications and variations of the present methods and compositions forenhancement of phytate phosphorus utilization and prevention ortreatment of tibial dyschondroplasia will be obvious to those skilled inthe art from the foregoing detailed description. Such modifications andvariations are intended to come within the scope of the appended claims.

I claim:
 1. A method for enhancing phytate phosphorus utilization in ananimal comprising administering to the animal an effective amount of avitamin D₃ derivative selected from the group consisting of1,25-dihydroxycholecalciferol;1,25-dihydroxy-26,27-hexadeuterocholecalciferol;1-hydroxycholecalciferol; 1,24,25-trihydroxycholecalciferol;1,25-dihydroxy-24-fluorocholecalciferol and mixtures thereof, incombination with animal feed containing 1.3% calcium by weight or lessand between 0.4 and 0.9% by weight phosphorus.
 2. The method of claim 1wherein the effective amount of vitamin D₃ derivative is between 1 and10 micrograms per kilogram feed.
 3. The method of claim 1 wherein thecalcium content of the feed is between 0.5 and 1.3% by weight.
 4. Themethod of claim 1 wherein further comprising administering an effectiveamount of active phytase to the animal to further enhance phytatephosphorus utilization.
 5. The method of claim 1 wherein the animal is apoultry animal.